Adipose-derived Mesenchymal Stem Cells Exhibit Tumor Tropism And Promote Tumorsphere Formation Of Breast Cancer Cells

Background and ObjectionBreast cancer remains one of the most frequent malignancies in women.Surgical treatment is currently the preferred option for almost all types of breast cancer.However,surgical incision procedures lead to the loss of breast volume and distortion of shape.Among the plastic surgery techniques currently available to reconstruct the breast,autologous fat grafting(AFG)is gaining major interest given its ease of harvest,low morbidity and capacity to improve tissue quality,particularly in breast-conserving surgery.With regards to the optimal technique for AFG,Yoshimura suggest that CAL(cell-assisted lipo-modelling)is effective for soft tissue augmentation and superior to conventional lipoinjection,in which the graft tissue is enriched with adipocyte-associated stem cells(ADSCs)which are increased by ex vivo culturing.However,the possibility that breast cancer cells might still reside in breast cancer patients after surgical treatment cannot be completely excluded.Transplantation of adipose tissue,consisting of ADSCs that are metabolically active and secrete various cytokines,does not simply behave as an inert filler and tends to influence the cancer microenvironment.Hence,it would be especially of interest to assess the oncological safety of cell-assisted lipo-modelling.The majority of current studies concerning the interaction between ADSCs and breast cancer cells were performed in a two-dimensional(2D)context in vitro,which did not fully simulate in vivo conditions.Hence,we established an in vitro 3D culture system and applied an in vivo animal model to investigate the interaction between ADSCs and MCF-7 breast cancer cells in tumor development in this study,mainly focusing on the tropism of ADSCs towards breast cancer cells and the potential mechanism of ADSCs on promoting MCF-7 cells progression.Materials and methods1.Cell preparation and identificationHuman ADSCs were isolated from abdominal liposuction aspirates of a healthy female patient.MCF-7 cells were obtained from Research Laboratory Collaboration Alliance of Nan Fang Hospital.2.Preparation of co-culture conditioned mediaTo study the effects of cytokines from a co-culture system on MCF-7 cells,ADSCs and MCF-7 co-culture conditioned media(AM-CM)was prepared.3.Cell membrane labeling and co-culture in MatrigelADSCs and MCF-7 cells were respectively stained with Vybrant(?)DiI Cell-Labeling Solution and DiO Cell-Labeling Solution.The same amounts of ADSCs and MCF-7 cells were mixed uniformly and seeded in Growth-factor-reduced(GFR)Matrigel.The interactions between ADSCs and MCF-7 cells were observed continuously in Matrigel for 96h using a confocal laser-scanning microscope.4.Scanning electron microscopy(SEM)For scanning electron microscopy,the same amounts of ADSCs and MCF-7 cells were co-cultured in Matrigel on round glass coverslips in 12-well plates.After 2 days,co-culture samples were fixed and examined under a scanning electron microscope.5.In vitro Transwell migration assayADSCs migration assays were performed using Transwell migration chambers.ADSCs were plated in the upper wells,whereas MCF-7 cells or AM-CM were dispensed in the lower chamber.Controls were represented by serum-free medium(DMEM).To further investigate the mechanisms underlying the cells migration in this co-culture system,MCF-7 cells were co-cultured with ADSCs cells in Matrigel.MCF-7 alone served as the control.Cells in both groups were collected for gene analysis at day 1,5,9.6.Tumor-bearing mice preparation and in vivo imaging of ADSCs homing to tumorsTumor-bearing models were prepared as previously described.We used cell tracer Vybrant(?)Dil to trace ADSCs in vivo.ADSCs were injected into the tail veins of tumor-bearing mice.Bioluminescence imaging was conducted to trace the ADSCs using an in vivo imaging system 4 weeks after the ADSCs injection.7.Tumorsphere formation in vitroTo investigate the tumorsphere formation capacity of MCF-7 cells under different circumstances in vitro,MCF-7 cells were either co-cultured with ADSCs or treated with 1ml AM-CM in Matrigel in 6-wells plates.MCF-7 cells alone served as a control.The diameters of tumorspheres in four random fields of each well were counted and photographed.To further investigate the effects of cytokines from a co-culture system on tumorsphere formation of MCF-7 cells,MCF-7 cells were treated with 1ml AM-CM in Matrigel in 6-wells plates.MCF-7 cells alone served as a control.On day 1,3 and 9,the Matrigel cultures were made into single-cell suspensions using Dispase and cells in both groups were collected for subsequent gene analysis.8.Xenograft assays in nude mice and frozen sectioningFor xenograft experiments,MCF-7 cells mixed with ADSCs were injected into the right groin adipose pad,whereas MCF-7 cells alone injected into the left groin adipose pad served as a control.Tumor volumes were measured using vemier caliper every 4 days for a consecutive 32 days and calculated as the formula:Tumor volume=lengthxwidth2/2,following previously published protocols.The mice were sacrificed at 40 days,and the tumor samples were then removed for further analysis.9.Quantitative real-time polymerase chain reaction(PCR)Quantitative real-time PCR was performed with primer-probe sequences for MIP-18,MIP-3?,E-Cadherin,Vimentin,SOX2 and OCT4 with previously designed primersfor the expression analysis.PCR specificity was assessed by the Ct method using ?-actin as an endogenous reference gene.10.Western blot analysisThe detection for E-Cadherin and Vimentin was performed with WesternBreeze Chemiluminescent Detection Kit.?-actin served as an internal control.11.Statistical analysisQuantitative results were expressed as mean± standard errors of the means ±SD.The comparison between co-culture or AM-CM treated samples at different time points with MCF-7 cells were examined for statistical significance using the Student's Mest with SPSS22.0 software.Multiple comparisons of migrated ADSCs in Transwell migration assay and tumorspheres diameter in in vitro tumorsphere formation assays were performed using one-way ANOVA.L-S-D post hoc analysis was performed to ADSCsertain significance between groups.A value of P<0.05 was considered statistically significant.Results1.Tumor tropism of ADSCs,chemokine expression and cell interactionsBoth ADSCs and MCF-7 cells were evenly distributed at 12h after co-culture.At 24h,ADSCs were touching and surrounding MCF-7 cells.ADSCs were observed inside MCF-7 turmorspheres at 96h after co-culture.In the Transwell system,the migration activity of ADSCs in upper wells was significantly promoted by MCF-7 cells in the lower chambers and,to a lesser extent,by the secreted cytokines from the co-culture system compared to the control.The expression levels of inflammatory chemokine macrophage inflammatory protein-1?(MIP-18)and MIP-3? in co-culture system(ADSCs+MCF-7)were markedly increased compared with the control(MCF-7/DMEM).To trace fluorescence-labeled ADSCs in mice,tumors and important organs were carefully dissected from mice and observed under living fluorescence system.The results revealed that fluorescence signal were mainly concentrated in tumor tissue and nearly no signal could be seen in other organs.The morphologic appearance of co-cultures of ADSCs and MCF-7 cells was observed by scanning electron microscopy.The spherical MCF-7 cells grew into tumorspheres with irregular surfaces in Matrigel substrate.Tumorspheres were surrounded by ADSCs,which was recognized by their flatly spread morphology.At higher magnifications,the construction of connections could be observed bridging the ADSCs and MCF-7 cells.2.ADSCs enhance tumorsphere formation,cancer stem cell(CSC)marker expression,and in vivo tumor formation of MCF-7 cellsTumorsphere formation was observed in our 3D culture system.MCF-7 cells co-cultured with ADSCs exhibited a significant capacity to form tumorspheres,whereas the AM-CM also exhibited a marked effect on tumorsphere formation.Quantitative analysis revealed that tumorspheres in co-culture groups(ADSCs+MCF-7)were larger than those treated with AM-CM(MCF-7/AM-CM).As a control,MCF-7 cells alone(MCF-7/DMEM)in Matrigel exhibited the weakest tumorsphere formation capacity.RNA expression analysis further revealed a higher expression of key CSC markers SOX2 and OCT4 in AM-CM treated MCF-7(MCF-7/AM-CM)than in the MCF-7 cells alone(MCF-7/DMEM).To further evaluate the effect of ADSCs on tumor growth of MCF-7 cells in vivo,we established a xenograft model in which MCF-7 cells were mixed with or without ADSCs and were then inoculated subcutaneously into nude mice.,the tumor volume of the ADSCS-treated group was dramatically increased compared with that of the control group.HE staining revealed that the necrotic area in ADSCS-treated tumor tissue was considerably reduced compared with that in controls.These results indicate that ADSCs may enhance the stemness expression and tumor-promoting properties of MCF-7 cells.3.Expression of epithelial and mesenchymal markersGene analysis revealed a reduced E-Cadherin(epithelial marker)expression and increased Vimentin(mesenchymal marker)expression in the AM-CM treated MCF-7 cells compared with the control MCF-7 cells.Protein analysis revealed a similar expression tendency of E-Cadherin and Vimentin in AM-CM treated MCF-7 cells and the control MCF-7 cells.ConclusionMCF-7 cells may exert a tropism effect on ADSCs,MIP-1? and MIP-3? may be involved in mediating this process.ADSCs promot MCF-7 cells epithelial-mesenchymal transition and stem-like cell transformation.ADSCs enhance MCF-7 cells tumorsphere formation in vivo and in vitro.